The enzymatic cleavage of double-stranded RNA is an essential step in the maturation and decay of many eukaryotic and prokaryotic RNAs. Double-stranded (ds) RNA cleavage is carried out by members of the ribonuclease Ill superfamily of endoribonucleases. RNase III orthologues participate in mRNA and rRNA maturation, mRNA degradation, and antisense RNA action. An important recent finding is the central involvement of the RNase III orthologue "Dicer" in RNA interference (RNAi). RNAi is proposed to suppress viral infection, inhibit retroposon movement, and participate in specific developmental pathways in eukaryotes, including humans. The occurrence of highly conserved sequences and structural elements indicate that RNase Ill orthologues use the same catalytic mechanism, which is unknown. The most studied member of the RNase III superfamily is Escherichia coli ribonuclease III and there has been recent significant progress in determining the E. coil RNase Ill mechanism of action. The long-range goal of this project is to determine the mechanism of dsRNA cleavage by E. coil RNase Ill. The specific aims of the project are to: (1) determine the functional roles of conserved amino acids in the catalytic domain. To accomplish this, RNase Ill mutants with substitutions of conserved catalytic domain residues will be tested in substrate binding and cleavage assays; (2), determine divalent metal ion stoichiometry and function in catalysis. To accomplish this, kinetic assays of cleavage, and metal ion binding assays using RNase Ill and selected mutants will be performed; (3), identify sequence and structural features in the RNase Ill double-stranded-RNA-binding domain (dsRBD) that confer binding energy and optimize catalytic efficiency. This will be accomplished by analyzing RNA binding of specific dsRBD mutants, and by phage display; (4), determine the stoichiometry of an RNase Ill-substrate complex, and identify intersubunit interactions important for RNase Ill function. Gel filtration, centrifugation, and substrate binding and cleavage assays using artificial heterodimers will be performed. Accomplishing these aims not only will provide important information on E. coil RNase Ill, but will also provide mechanistic insight on RNase Ill orthologue function in eukaryotic RNA processing and degradation pathways, including RNAi.